Sperm motility is essential for successful fertilization, with hyperactivated motility (HA) playing a critical role in sperm migration and penetration of the oocyte's protective layers. HA is regulated by intracellular Ca2+ influx through the sperm-specific CatSper channel. While CatSper activation is known to be influenced by various chemical factors, its sensitivity to physical stimuli such as temperature remain unclear. In this study, we investigated the impact of temperature on CatSper activity and HA in human sperm. The percentage of sperm exhibiting HA increased significantly in a temperature-dependent manner without affecting total motility or viability. Temperature-induced HA was dependent on CatSper-mediated Ca2+ influx, as pharmacological inhibition of CatSper abolished the response. Spectrofluorometric assays and patch-clamp experiments revealed that higher temperatures modestly but consistently enhanced CatSper activation as well as other capacitation-associated cAMP/PKA signaling pathways. These findings demonstrate that rather than acting as a dedicated thermosensor, CatSper displays physiologically relevant temperature-dependent modulation, suggesting a role for thermal cues in sperm capacitation and motility within the female reproductive tract.

BackgroundChloride ions (Cl) regulate sperm physiology, influencing spermatogenesis, volume regulation, capacitation, and fertilization processes. They contribute to the maintenance of membrane potential and intracellular pH, both of which are critical for sperm motility and capacitation. Any deviation in Cl homeostasis causes impaired sperm function and male infertility. Although several Cl channels and transporters have been implicated in the Cl homeostasis and osmoregulation of sperm cells, the precise mechanisms and molecular components governing volume regulation during sperm development remain unclear.MethodsWe used a combination of electrophysiological recordings via the patch-clamp technique and biochemical analyses, including western blotting and immunocytochemistry, to demonstrate the functional expression of a novel chloride channel, Chloride Intracellular Channel 4 (CLIC4), in the plasma membrane of sperm cells. To assess physiological roles, we analyzed sperm cells from wild type and null mutant mice (clic4-/-), measuring motility, morphology and acrosome reaction.ResultsWe identified previously uncharacterized IAA-94-sensitive chloride currents in mouse sperm cells. Genetic ablation of CLIC4 eliminated these IAA-94-sensitive currents. Notably, CLIC4 regulates cell volume during sperm maturation without altering membrane potential, motility, or the acrosome reaction. CLIC4 activity in sperm cells is modulated by Protein Kinase C (PKC).ConclusionCLIC4 is a key component of the sperm cell volume regulation machinery, modulating Cl fluxes during maturation. These findings provide new insights into the molecular basis of sperm osmoregulation and may inform therapeutic strategies for male infertility.

The study of human sperm motility has been a topic of interest for decades due to its crucial role in fertility and reproductive health. While most analyses rely on 2D+t imaging of head trajectories, sperm naturally swim in three dimensions (3D), driven by complex flagellar motion. However, the lack of comprehensive 3D+t datasets has limited progress in this field. To address this, we present 3D-SpermFlagella, the first large-scale 3D+t dataset of human sperm flagellum centerline annotations. This dataset contains 135 tracked and annotated sperm, derived from our previously published multifocal video microscopy dataset 3D-SpermVid. Each flagellar centerline was annotated over time in three dimensions, incubated under non-capacitating (NCC) and capacitating (CC) conditions. The (x,y,z) coordinates are provided in both micrometers and voxels, making 3D-SpermFlagella a valuable resource for studying sperm motility in its full spatial complexity and for the development and benchmarking of AI-based models for tracking and segmentation. In this paper, we describe the segmentation and tracking methods, as well as the conditions and structure of the dataset.

After ejaculation, mammalian spermatozoa are not capable of fertilizing a metaphase II-arrested egg. They require to undergo a series of biochemical and physiological processes collectively known as capacitation. In all these processes, the regulation of calcium ions fluxes plays essential roles and involves participation of many channels and transporters localized in the plasma membrane as well as in the membrane of intracellular organelles. In mammalian sperm, a fraction of these molecules has been proposed to contribute to mature sperm function. However, in many cases, the evidence for the presence of a given protein is based on the use of agonists and antagonists with more than one target. In this review, we will critically analyze the published evidence supporting the presence of these molecules in mammalian sperm with special emphasis to methods involving tandem mass spectrometry identification, electrophysiological evidence and controlled immunoassays.

Sperm capacitation involves a series of biochemical and physiological changes essential for fertilization. A critical regulator of capacitation, the soluble adenylyl cyclase (sAC; ADCY10)-dependent production of the second messenger cyclic AMP (cAMP), drives key downstream events such as protein kinase A (PKA) substrate phosphorylation. sAC activity is directly stimulated by bicarbonate (HCO3 -) and calcium (Ca2+). CatSper, a sperm-specific Ca2+ channel, is considered the primary pathway for Ca2+ influx during capacitation; however, emerging evidence suggests additional pathways exist. This study reveals that bovine serum albumin (BSA) influences the dynamics of intracellular Ca2+ concentration ([Ca2+]i) in CatSper1 knockout (KO) sperm and plays a novel role in sAC activation. Using single-cell live imaging and flow cytometry, we observed a rapid [Ca2+]i rise in the head of CatSper1 KO sperm under capacitating conditions, indicating an alternative Ca2+ entry mechanism. BSA alone, in the absence of HCO3 -, triggered a significant [Ca2+]i rise. Removal of extracellular Ca2+ abolished this [Ca2+]i rise, confirming the necessity of Ca2+ influx. This BSA-induced [Ca2+]i rise was upstream of sAC activation, since it was not affected by sAC inhibitors and led to increased cAMP production and PKA substrate phosphorylation. Our findings provide new insights into the regulatory mechanisms of sAC, highlighting the existence of a CatSper-independent Ca2+ entry pathway activated by BSA during sperm capacitation. This rapid [Ca2+]i rise is initiated in the sperm head and propagates throughout the cell, and is sufficient to activate sAC and stimulate cAMP synthesis independently of HCO3 -. KEY POINTS: Sperm capacitation, essential for fertilization, is regulated by sAC, which produces cAMP in response to HCO3 - and Ca2+, driving key events like protein kinase A substrate phosphorylation. We demonstrate the existence of a CatSper-independent Ca2+ entry pathway that initiates in the sperm head and propagates throughout the cell, occurring rapidly after sperm encounters albumin, a critical component of the capacitation medium used in in vitro fertilization procedures in mammals. This albumin-induced Ca2+ influx is sufficient to activate sAC and stimulate cAMP synthesis independently of HCO3 -. We further reveal a novel role for albumin, beyond its well-established function as a cholesterol acceptor, in triggering this rapid Ca2+ influx and downstream signalling events essential for sperm capacitation. By demonstrating a CatSper-independent regulatory pathway, we expand the current paradigm of Ca2+ signalling in sperm physiology.

Accurate determinations of acrosome reaction (AR) are fundamental to obtain physiologically relevant information of this essential event for sperm fertilization. For decades, the AR was exclusively studied in fixed preparation; more recently, new methods were developed to evaluate the AR in live sperm and in real time. These new technologies involved the use of genetically modified mice models, dyes with ion affinity, and fluorescent microscopy. In addition, these techniques allowed the reproduction field the possibility to follow the AR directly in the female tract under physiological conditions. Despite these advances, maintaining transgenic colonies is expensive for most research laboratories. Here, we present a methodology which can be mounted in a simple epifluorescence microscopy set in combination with appropriate filters enabling to follow the AR in real time. The implemented technique allows also to follow the AR in combination with [Ca2+]i, an ion directly involved in the regulation of this exocytotic reaction. In this chapter, we present in a simple way the methodology required to obtain AR/[Ca2+]i determinations using Fluo-4 and FM4-64 fluorescence recordings in live sperm under physiological and non-physiological stimuli. Also, we explain step by step the analysis and treatment to the images obtained during the recordings to determine AR quantitative information. The general methodology can be applied to measure other sperm parameters for which fluorescent dyes are available, such as intracellular pH (pHi) in combination with [Ca2+]i and/or the AR.

Understanding human fertility requires dynamic and three-dimensional (3D) analysis of sperm movement, which extends beyond the capabilities of traditional datasets focused primarily on two-dimensional sperm motility or static morphological characteristics. To address this limitation, we introduce the 3D+t Multifocal Imaging Dataset of Human Sperm (3D-SpermVid), a repository comprising 121 multifocal video-microscopy hyperstacks of freely swimming sperm cells, incubated under non-capacitating conditions (NCC) and capacitating conditions (CC). This collection enables detailed observation and analysis of 3D sperm flagellar motility patterns over time, offering novel insights into the capacitation process and its implications for fertility. Data were captured using a multifocal imaging (MFI) system based on an optical microscope equipped with a piezoelectric device that adjusts focus at various heights, recording sperm movement in a volumetric space. By making this data publicly available, we aim to enable applications in deep learning and pattern recognition to uncover hidden flagellar motility patterns, fostering significant advancements in understanding 3D sperm morphology and dynamics, and developing new diagnostic tools for assessing male fertility, as well as assisting in the self-organizaton mechanisms driving spontaneous motility and navigation in 3D.

The fractal dimension of human sperm flagellar curves is used to look up for features pointing to hyperactivation movement. The original 2D fractal dimension formula is directly extended to calculate its 3D counterpart, by means of the Katz Fractal Dimension formula. For each spermatozoon a time-distribution of the flagellar fractal dimension is produced. A distribution is obtained to analyze the time-mean flagellar fractal dimension, and two clear clusters appear that hints to some sperm cells having hyperactivated movement.

Dave Garbers' work significantly contributed to our understanding of sperm's regulated motility, capacitation, and the acrosome reaction. These key sperm functions involve complex multistep signaling pathways engaging numerous finely orchestrated elements. Despite significant progress, many parameters and interactions among these elements remain elusive. Mathematical modeling emerges as a potent tool to study sperm physiology, providing a framework to integrate experimental results and capture functional dynamics considering biochemical, biophysical, and cellular elements. Depending on research objectives, different modeling strategies, broadly categorized into continuous and discrete approaches, reveal valuable insights into cell function. These models allow the exploration of hypotheses regarding molecules, conditions, and pathways, whenever they become challenging to evaluate experimentally. This review presents an overview of current theoretical and experimental efforts to understand sperm motility regulation, capacitation, and the acrosome reaction. We discuss the strengths and weaknesses of different modeling strategies and highlight key findings and unresolved questions. Notable discoveries include the importance of specific ion channels, the role of intracellular molecular heterogeneity in capacitation and the acrosome reaction, and the impact of pH changes on acrosomal exocytosis. Ultimately, this review underscores the crucial importance of mathematical frameworks in advancing our understanding of sperm physiology and guiding future experimental investigations.

Mammalian sperm delve into the female reproductive tract to fertilize the female gamete. The available information about how sperm regulate their motility during the final journey to the fertilization site is extremely limited. In this work, we investigated the structural and functional changes in the sperm flagellum after acrosomal exocytosis (AE) and during the interaction with the eggs. The evidence demonstrates that the double helix actin network surrounding the mitochondrial sheath of the midpiece undergoes structural changes prior to the motility cessation. This structural modification is accompanied by a decrease in diameter of the midpiece and is driven by intracellular calcium changes that occur concomitant with a reorganization of the actin helicoidal cortex. Midpiece contraction occurs in a subset of cells that undergo AE, and live-cell imaging during in vitro fertilization showed that the midpiece contraction is required for motility cessation after fusion is initiated. These findings provide the first evidence of the F-actin network’s role in regulating sperm motility, adapting its function to meet specific cellular requirements during fertilization, and highlighting the broader significance of understanding sperm motility.

The flagellar movement of the mammalian sperm plays a crucial role in fertilization. In the female reproductive tract, human spermatozoa undergo a process called capacitation which promotes changes in their motility. Only capacitated spermatozoa may be hyperactivated and only those that transition to hyperactivated motility are capable of fertilizing the egg. Hyperactivated motility is characterized by asymmetric flagellar bends of greater amplitude and lower frequency. Historically, clinical fertilization studies have used two-dimensional analysis to classify sperm motility, despite the inherently three-dimensional (3D) nature of sperm motion. Recent research has described several 3D beating features of sperm flagella. However, the 3D motility pattern of hyperactivated spermatozoa has not yet been characterized. One of the main challenges in classifying these patterns in 3D is the lack of a ground-truth reference, as it can be difficult to visually assess differences in flagellar beat patterns. Additionally, it is worth noting that only a relatively small proportion, approximately 10-20% of sperm incubated under capacitating conditions exhibit hyperactivated motility. In this work, we used a multifocal image acquisition system that can acquire, segment, and track sperm flagella in 3D+t. We developed a feature-based vector that describes the spatio-temporal flagellar sperm motility patterns by an envelope of ellipses. The classification results obtained using our 3D feature-based descriptors can serve as potential label for future work involving deep neural networks. By using the classification results as labels, it will be possible to train a deep neural network to automatically classify spermatozoa based on their 3D flagellar beating patterns. We demonstrated the effectiveness of the descriptors by applying them to a dataset of human sperm cells and showing that they can accurately differentiate between non-hyperactivated and hyperactivated 3D motility patterns of the sperm cells. This work contributes to the understanding of 3D flagellar hyperactive motility patterns and provides a framework for research in the fields of human and animal fertility.

As in most cells, intracellular pH regulation is fundamental for sperm physiology. Key sperm functions like swimming, maturation, and a unique exocytotic process, the acrosome reaction, necessary for gamete fusion, are deeply influenced by pH. Sperm pH regulation, both intracellularly and within organelles such as the acrosome, requires a coordinated interplay of various transporters and channels, ensuring that this cell is primed for fertilization. Consistent with the pivotal importance of pH regulation in mammalian sperm physiology, several of its unique transporters are dependent on cytosolic pH. Examples include the Ca2+ channel CatSper and the K+ channel Slo3. The absence of these channels leads to male infertility. This review outlines the main transport elements involved in pH regulation, including cytosolic and acrosomal pH, that participate in these complex functions. We present a glimpse of how these transporters are regulated and how distinct sets of them are orchestrated to allow sperm to fertilize the egg. Much research is needed to begin to envision the complete set of players and the choreography of how cytosolic and organellar pH are regulated in each sperm function.

In the realm of 3D image processing, accurately representing the geometric nuances of line curves is crucial. Building upon the foundation set by the slope chain code, which adeptly represents intricate two-dimensional curves using an array capturing the exterior angles at each vertex, this study introduces an innovative 3D encoding method tailored for polygonal curves. This 3D encoding employs parallel slope and torsion chains, ensuring invariance to common transformations like translations, rotations, and uniform scaling, while also demonstrating robustness against mirror imaging and variable starting points. A hallmark feature of this method is its ability to compute tortuosity, a descriptor of curve complexity or winding nature. By applying this technique to biomedical engineering, we delved into the flagellar beat patterns of human sperm. These insights underscore the versatility of our 3D encoding across diverse computer vision applications.

We investigate the dynamics and hydrodynamics of a human spermatozoa swimming freely in 3D. We simultaneously track the sperm flagellum and the sperm head orientation in the laboratory frame of reference via high-speed high-resolution 4D (3D+t) microscopy, and extract the flagellar waveform relative to the body frame of reference, as seen from a frame of reference that translates and rotates with the sperm in 3D. Numerical fluid flow reconstructions of sperm motility are performed utilizing the experimental 3D waveforms, with excellent accordance between predicted and observed 3D sperm kinematics. The reconstruction accuracy is validated by directly comparing the three linear and three angular sperm velocities with experimental measurements. Our microhydrodynamic analysis reveals a novel fluid flow pattern, characterized by a pair of vortices that circulate in opposition to each other along the sperm cell. Finally, we show that the observed sperm counter-vortices are not unique to the experimental beat, and can be reproduced by idealised waveform models, thus suggesting a fundamental flow structure for free-swimming sperm propelled by a 3D beating flagellum.

The sperm ability to fertilize involves the regulation of ATP levels. Because inside cells, ATP is complexed with Mg2+ ions, changes in ATP levels result in changes in intracellular Mg2+ concentration ([Mg2+]i), which can be followed using intracellular Mg2+ sensors such as Mag-520. In this work, we tested conditions known to decrease sperm ATP such as starvation and capacitation. As expected, in these conditions [Mg2+]i increased in all cell compartments. In contrast, when ATP increases, such as adding nutrients to starved sperm, [Mg2+]i significantly decreases in all compartments. On the other hand, when the acrosome reaction was induced, either with progesterone or with ionomycin, [Mg2+]i was differentially regulated in the head and mid-piece. While Mag-520 fluorescence increased in the sperm mid-piece, it decreased in the head. These changes were observed in capacitated as well as in starved sperm but not in sperm incubated in conditions that do not support capacitation. Changes in [Mg2+]i were still observed when the sperm were incubated in high extracellular Mg2+ suggesting that this decrease is not due to Mg2+ efflux. Interestingly, the progesterone and ionomycin effects on [Mg2+]i were abolished on sperm incubated in Ca2+-free media. Altogether, these results indicate that [Mg2+]i is regulated in sperm during capacitation and acrosomal reaction, and suggest that these measurements can serve to evaluate ATP levels in real time.

Sperm capacitation is a complex process that takes place in the female reproductive tract and empowers mammalian sperm with the competence to fertilize an egg. It consists of an intricate cascade of events that can be mimicked in vitro through incubation in a medium containing essential components, such as bicarbonate, albumin, Cat+, and energy substrates, among others. Genetic and pharmacological studies have underscored the unique significance of the K+ channel SLO3 in membrane potential hyperpolarization, as evidenced by the infertility of mice lacking its expression. Notably, two key molecular events, sperm hyperpolarization and intracellular alkalinization, are central to the capacitation process. SLO3 is activated by alkalinization. However, the molecular mechanisms responsible for intracellular alkalization and activation of SLO3 are not completely understood. In this study, we examined the impact of Na+/H+ exchangers (NHEs) on mouse sperm membrane hyperpolarization during capacitation. Pharmacological inhibition of the NHE1 blocked membrane hyperpolarization. A similar effect was observed in sperm deficient of the Cat+ channel CatSper because of NHE1 not being activated by Cat+. In addition, the sperm-specific NHE (sNHE) KO did not show membrane hyperpolarization upon capacitation or induction with cAMP analogs. Our results show that sNHE is dually modulated by cAMP and membrane hyperpolarization probably through its cyclic nucleotide- binding domain and the voltage-sensor motif, respectively. Together, sNHE and NHE1 provide the alkalinization need for SLO3 activation during capacitation.

To become fertile, mammalian sperm are required to undergo capacitation in the female tract or in vitro in defined media containing ions (e.g. HCO3 -, Ca2+, Na+, and Cl-), energy sources (e.g. glucose, pyruvate) and serum albumin (e.g. bovine serum albumin (BSA)). These different molecules initiate sequential and concomitant signaling pathways, leading to capacitation. Physiologically, capacitation induces changes in the sperm motility pattern (e.g. hyperactivation) and prepares sperm for the acrosomal reaction (AR), two events required for fertilization. Molecularly, HCO3 - activates the atypical adenylyl cyclase Adcy10 (aka sAC), increasing cAMP and downstream cAMP-dependent pathways. BSA, on the other hand, induces sperm cholesterol release as well as other signaling pathways. How these signaling events, occurring in different sperm compartments and with different kinetics, coordinate among themselves is not well established. Regarding the AR, recent work has proposed a role for glycogen synthase kinases (GSK3α and GSK3β). GSK3α and GSK3β are inactivated by phosphorylation of residues Ser21 and Ser9, respectively, in their N-terminal domain. Here, we present evidence that GSK3α (but not GSK3β) is present in the anterior head and that it is regulated during capacitation. Interestingly, BSA and HCO3 - regulate GSK3α in opposite directions. While BSA induces a fast GSK3α Ser21 phosphorylation, HCO3 - and cAMP-dependent pathways dephosphorylate this residue. We also show that the HCO3--induced Ser21 dephosphorylation is mediated by hyperpolarization of the sperm plasma membrane potential (Em) and by intracellular pH alkalinization. Previous reports indicate that GSK3 kinases mediate the progesterone-induced AR. Here, we show that GSK3 inhibition also blocks the Ca2+ ionophore ionomycin-induced AR, suggesting a role for GSK3 kinases downstream of the increase in intracellular Ca2+ needed for this exocytotic event. Altogether, our data indicate a temporal and biphasic GSK3α regulation with opposite actions of BSA and HCO3 -. Our results also suggest that this regulation is needed to orchestrate the AR during sperm capacitation.

In addition to its central role in cellular metabolism, adenosine 5′-triphosphate (ATP) is an important extracellular signalling molecule involved in various physiological processes. In reproduction, extracellular ATP participates in both autocrine and paracrine paths regulating gametogenesis, gamete maturation and fertilisation. This review focusses on how extracellular ATP modulates sperm physiology with emphasis on the mammalian acrosome reaction. The presence of extracellular ATP in the reproductive tract is primarily determined by the ion channels and transporters that influence its movement within the cells comprising the tract. The main targets of extracellular ATP in spermatozoa are its own transporters, particularly species-specific sperm purinergic receptors. We also discuss notable phenotypes from knock-out mouse models and human Mendelian inheritance related to ATP release mechanisms, along with immunological, proteomic, and functional observations regarding sperm purinergic receptors and their involvement in sperm signalling.

Image-Based Flow Cytometry (IBFC) is a potent tool for the detailed analysis and quantification of cells in intricate samples, facilitating a comprehensive understanding of biological processes. This study leverages the ResNet50 model to address IBFC's object-defocusing issue, an inherent challenge when imaging a 3D object with stationary optics. A dataset of 604 mouse sperm IBFC images (both bright field and fluorescence) underpins the exceptional capability of the ResNet50 model to reliably identify optimally focused images of the sperm head and flagella (F1-Score of 0.99). A U-Net model was subsequently employed to accurately segment the sperm head and flagellum in images selected by ResNet50. Notably, the flagellum presents a significant challenge due to its sub-diffraction transversal dimensions of 0.4 to 1 micrometers, resulting in minimal light intensity gradients. The U-Net model, however, demonstrates exceptional efficacy in precisely segmenting the flagellum and head (dice = 0.81). The combined ResNet50/U-Net approach offers significant promise for enhancing the efficiency and reliability of sperm analysis via IBFC, and could potentially drive advancements in reproductive research and clinical applications. Additionally, these innovative strategies may be adaptable to the analysis of other cell types.

The acrosome is a lysosome-related vesicular organelle located in the sperm head. The acrosomal reaction (AR) is an exocytic process mediated by Ca2+ and essential for mammalian fertilization. Recent findings support the importance of acrosomal alkalinization for the AR. Mibefradil (Mib) and NNC 55-0396 (NNC) are two amphipathic weak bases that block the sperm-specific Ca2+ channel (CatSper) and induce acrosomal pH (pH alpha) increase by accumulating in the acrosomal lumen of mammalian sperm. This accumulation and pH alpha elevation increase the intracellular Ca2+ concentration ([Ca2+](i)) and trigger the AR by unknown mechanisms of Ca2+ transport. Here, we investigated the pathways associated with the pH alpha increase-induced Ca2+ signals using mouse sperm as a model. To address these questions, we used single-cell Ca2+ imaging, the lysosomotropic agent Gly-Phe-beta-naphthylamide (GPN) and pharmacological tools. Our findings showthat Mib andNNCincrease pH alpha and release acrosomal Ca2+ without compromising acrosomal membrane integrity. Our GPN results indicate that the osmotic component does not significantly contribute to acrosomal Ca2+ release caused by pH alpha rise. Inhibition of two-pore channel 1 (TPC1) channels reduced the [Ca2+](i) increase stimulated by acrosomal alkalinization. In addition, blockage of Ca2+ release-activated Ca2+ (CRAC) channels diminished Ca2+ uptake triggered by pH alpha alkalinization. Finally, our findings contribute to understanding how pH alpha controls acrosomal Ca2+ entry during AR inmouse sperm.